Proteins that selectively bind to selected targets by way of non-covalent interaction play a crucial role as reagents in biotechnology, medicine, bioanalytics as well as in the biological and life sciences in general. Antibodies, i.e. immunoglobulins, are a prominent example of this class of proteins. Despite the manifold needs for such proteins in conjunction with recognition, binding and/or separation of ligands/targets, almost exclusively immunoglobulins are currently used.
Additional proteinaceous binding molecules that have antibody-like functions are certain members of the lipocalin family, which have naturally evolved to endogenously bind ligands. Lipocalins occur in many organisms, including vertebrates, insects, plants and bacteria. Members of the lipocalin protein family (Pervaiz, S., & Brew, K. (1987) FASEB J. 1, 209-214) are typically small, secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular-recognition properties: their ability to bind various, principally hydrophobic molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants), their binding to specific cell-surface receptors and their formation of macromolecular complexes. Although they have, in the past, been classified primarily as transport proteins, it is now clear that the lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and the synthesis of prostaglandins. The lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homeostasis (reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14 and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).
α1-Microglobulin (α1m)—also known as protein HC, α1-glycoprotein or α1-microglyco-protein—is a 26 kDa glycoprotein with 184 amino acid residues which is abundant in blood plasma, urine, and connective tissue of humans as well as vertebrate animals (Åkerström, B., Lögdberg, L., Berggärd, T., Osmark, P., and Lindqvist, A. (2000) Biochim Biophys Acta 1482, 172-184). Based on characteristic amino acid sequence motifs, α1m has been assigned a member of the lipocalin family (Pervaiz, S., and Brew, K. (1987) FASEB J 1, 209-214; Pervaiz, S., and Brew, K. (1985) Science 228, 335-337), although this has been done without knowing the three-dimensional structure of this biomolecule. Natural a1m from urine and plasma is heterogeneous in size and charge and has a characteristic yellow-brown color Åkerström, B., and Berggärd, I. (1979) Eur J Biochem 101, 215-223; Berggärd, T., Cohen, A., Persson, P., Lindqvist, A., Cedervall, T., Silow, M., Thogersen, I. B., Jonsson, J. A., Enghild, J. J., and Åkerström, B. (1999) Protein Sci 8, 2611-2620), an attribute that also served to designate this lipocalin (kerström, B., Lögdberg, L., Berggärd, T., Osmark, P., and Lindqvist, A. (2000) Biochim Biophys Acta 1482, 172-184). α1m is glycosylated at three sites: two complex carbohydrates are N-linked to residues Asn17 and Asn96 while Thr5 is O-glycosylated (Ekström, B., Lundblad, A., and Svensson, S. (1981) Eur J Biochem 114, 663-666; Escribano, J., Lopex-Otin, C., Hjerpe, A., Grubb, A., and Mendez, E. (1990) FEBS Lett 266, 167-170). Since its initial identification in humans (Ekström, B., Peterson, P. A., and I., B. (1975) Biochem Biophys Res Commun. 65, 1427-1433), α1m has been associated with various physiological processes including immunosuppression (Åkerström, B., Lögdberg, L., Berggärd, T., Osmark, P., and Lindqvist, A. (2000) Biochim Biophys Acta 1482, 172-184; Lögdberg, L., and Åkerström, B. (1981) Scand J Immunol 13, 383-390), lymphocyte stimulation, and also inhibition of lymphocyte cell proliferation (Wester, L., Michaelsson, E., Holmdahl, R., Olofsson, T., and Åkerström, B. (1998) Scand J Immunol 48, 1-7) as well as neutrophil chemotaxis (Mendez, E., Fernandez-Luna, J. L., Grubb, A., and Leyva-Cobian, F. (1986) Proc Natl Acad Sci USA 83, 1472-1475). Furthermore, several biochemical activities have been ascribed to α1m, in particular in the context of heme and tryptophan metabolism (Olsson, M. G., Olofsson, T., Tapper, H., and Åkerström, B. (2008) Free Radic Res 42, 725-736; Allhorn, M., Berggärd, T., Nordberg, J., Olsson, M. L., and Åkerström, B. (2002) Blood 99, 1894-1901) and with regard to reductase and radical scavenging functions (Allhorn, M., Klapyta, A., and Åkerström, B. (2005) Free Radic Biol Med 38, 557-567; Åkerström, B., Maghzal, G. J., Winterbourn, C. C., and Kettle, A. J. (2007) J Biol Chem 282, 31493-31503). In addition, α1m serves as an important biomarker in clinical diagnostics for the monitoring of renal tubular dysfunction, renal toxicity, preeclampsia, and hepatitis E (Bolt, H. M., Lammert, M., Selinski, S., and Bruning, T. (2004) Int Arch Occup Environ Health 77, 186-190; Taneja, S., Sen, S., Gupta, V. K., Aggarwal, R., and Jameel, S. (2009) Proteome Sci 7, 39; Yu, H., Yanagisawa, Y., Forbes, M. A., Cooper, E. H., Crockson, R. A., and MacLennan, I. C. (1983) J Clin Pathol 36, 253-259; Devarajan, P., Krawczeski, C. D., Nguyen, M. T., Kathman, T., Wang, Z., and Parikh, C. R. (2010) Am J Kidney Dis 56, 632-642; Anderson, U. D., Olsson, M. G., Rutardottir, S., Centlow, M., Kristensen, K. H., Isberg, P. E., Thilaganathan, B., Åkerström, B., and Hansson, S. R. (2011) Am J Obstet Gynecol 204, 520 e521-525). Apart from these observations, no dedicated physiological ligand for the central hydrophobic pocket of α1m—a characteristic feature of all lipocalin proteins (Flower, D. R. (1996) Biochem J 318, 1-14; Skerra, A. (2000) Biochim Biophys Acta 1482, 337-350)—could be identified so far.
Various PCT publications (e.g., WO 99/16873, WO 00/75308, WO 03/029463, WO 03/029471 and WO 2005/19256) disclose how muteins of various lipocalins (e.g. tear lipocalin and hNGAL lipocalin) can be constructed to exhibit a high affinity and specificity against a target that is different than a natural ligand of a wild type lipocalin. This can be done by mutating certain positions of the lipocalin in a rational manner.
Despite the advances made with certain lipocalins in terms of re-engineering their specificity, there remains a need for the generation of other lipocalin muteins that contain different binding sites and alternative lipocalin scaffolds that can be used for such generation. In view of the various potential applications for ligand- or target-binding proteins in the field of life sciences and biotechnology, the generation of muteins of yet other lipocalins would be desirable to, e.g., widen the spectrum of clinical targets against which lipocalin muteins may bind. Without knowing the secondary and tertiary structure of a lipocalin and hence its potential binding sites, any rational attempt to generate one or more muteins of that lipocalin to bind a target of interest would be futile.
Accordingly, to meet said need, the present disclosure provides, for example, the structural elucidation of the lipocalin scaffold human α1m to create, e.g., a collection (i.e. library) of lipocalin muteins including members (i.e. muteins) that have binding affinity and specificity to at least one target other than a target to which wild-type a1m binds. The present disclosure also provides lipocalin muteins whose pocket or loop region may contain more than one binding site as, e.g., the central pocket (i.e. cavity) of the a1m polypeptide is wider than that in other lipocalins.